More than 400,000 arthroplasties are performed annually in the US and up to 20 percent of these require revision surgery due to aseptic loosening. This complication constitutes a major source of morbidity and represents billions of dollars in health care costs. This proposal is designed to (1) define the role of TNFalpha/NFkB/RANK signal transduction in the disease process; and (2) develop a gene therapy protocol targeted to block the TNFalpha/NFkB/RANK mediated inflammatory bone resorption as a potential treatment for aseptic loosening. The hypothesis to be studied is that (i) particles stimulate NFkB activation leading to induction of TNFalpha; (ii) TNFalpha amplifies the inflammatory response as a paracrine mediator of NFkB stimulation; (iii) macrophage-secreted inflammatory mediators stimulate RANK ligand secretion in stromal cells; and (iv) RANK ligand stimulates osteoclastogenesis and bone resorption. It is the applicant group's hypothesis that TNFR-Ig primarily inhibits inflammation and directly inhibits osteoclastogenesis and bone resorption, while RANK-Ig/OPG are direct regulators of osteoclast formation, activation and apoptosis. These hypotheses will be investigated in a series of in vitro and in vivo experiments that include examination of the effects of particles on NFkB activation and cytokine secretion in cell lines genetically engineered to have deficient NFkB signaling, as well as in cells treated with TNFR-Ig and RANK-Ig. The effect of these proteins on macrophage induced (i) stromal cell expression of RANK ligand and OPG; (ii) osteoclastogenesis; and (iii) bone resorption will be assessed. Finally, they will investigate adeno-associated viral (AAV) gene transfer as an effective delivery system for TNFR-Ig and OPG. The applicants hypothesize that particles will stimulate AAV second strand synthesis and lead to target protein expression that will be sustained and occur after a period of dormancy. The hypothesis is that the AAV-transduced target proteins, TNFR-Ig and OPG, inhibit particle-induced inflammatory bone resorption in vivo, expecting that the effects will be greatest when used in combination, due to their actions at different stages in the process of inflammatory bone resorption.